Separative harvesting device

ABSTRACT

A harvesting device for capturing a biological product directly by binding the secreted biological product with a resin, discarding the nutrient medium and eluting the biological product as a concentrated solution, eliminating the steps of sterile filtration and volume reduction, thus allowing one to combine the steps of recombinant expression and separation of a biological product. The method allows loading of resin for column-purification, eliminating all steps of perfusion process and maintaining a sink condition of a toxic product in nutrient medium to optimize productivity of host cells. The instant invention also allows harvesting of solubilized inclusion bodies after the cells have been lysed and refolding of proteins inside the bioreactor.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.13/092,955, filed on Apr. 24, 2011, entitled SEPARATIVE BIOREACTOR, theentire contents of which is incorporated herein by reference.

FIELD OF INVENTION

The instant invention relates to a bioreactor design intended to captureand purify biological products within the bioreactor.

BACKGROUND OF THE INVENTION

The present invention relates to a novel bioreactor design forexpressing and separating a biological product from other components ina bioreaction borth, which combines the step of expressing andseparating within the bioreactor by binding the biological product witha resin within a bioreactor, discarding the nutrient medium and elutingthe biological product as a concentrated solution; this allowselimination at least two steps in the separation and purification ofbiological products—filtration or centrifugation to remove cell cultureand ultrafiltration for volume reduction—and possibly three steps,including loading of biological products on the purification columns.

For products which are expressed as inclusion bodies, the instantinvention allows cell lysis, inclusion body solubilization and proteinrefolding within the bioreactor.

The instant invention significantly reduces the process time and costwhile enhancing the yield by reducing degradation of biological productsduring manufacturing. Additional benefits of the instant inventioninclude avoiding perfusion process and reducing toxicity of theexpressed biological products to cell culture. No such invention existsin the prior art of bioreactors.

Downstream processing involves steps for cleaning up crude biologicalproducts to yield high purity products. Traditionally, these stepsinvolve using chromatography columns packed with highly specializedresings to capture and purify the desired biological products by theprocess of elution. With an exponential rise in the number of biologicalproducts being developed and marketed, there have been remarkabledevelopments in the field of downstream processing. These developmentshave however not caught up with the developments in the upstreamprocessing. A few years ago, an yield of 0.25 G of biological productper liter expressed by CHO cells was considered very high. Today, we arehovering yields around 10 G/L making it possible to accumulate a verylarge quantity of biological products, particularly as the sizes ofbioreactors have increased to thousands of liters.

There are three steps that connect the upstream and downstreamprocessing. First, the culture media must be filtered using fine filters(e.g., 0.22 microns) to remove cells (CHO cells have average size of 5microns). This step utilizes an array of filters since the cells arelikely to choke the filter surface easily and also require installingvessels that would receive the filtrate. This requires vessels ofthousands of liters of capacity to match the size of the bioreactors.The next step is the reduction of the volume of filtrate since it is notpossible to load such large volumes on columns that have limited flowrate. This is the stage where most often a cross-flow type filtration isused, again with a large bank of filters to complete the concentrationprocess as quickly as possible.

The mechanism of cross flow filtration places severe pressure on thesolution and causes breakdown and precipitation of biological productsresulting in losses of generally 10-20% at this stage. Both of theseprocesses take a very long time and during this processing it is notpossible to keep the biological product solution at a lower temperatureresulting in the degradation of biological product as well.

The third step is to load the concentrated solution in a chromatographycolumn containing a binding media, a specific resin with affinity forthe target biological product. Even though the volume of liquid has beenreduced considerably at this stage, the loading steps, nevertheless,take substantial time to complete the loading.

The time and cost-consuming steps of filtration, chromatography andpurification slow down the manufacturing process and add substantialcapital cost requirement to establish cGMP-grade manufacturingoperations.

Bioreactors used in the upstream processing are vessels that allowgrowth of cell culture to express biological products and for reasonshistoric and traditional, a clear demarcation line exists between theexpression of biological product and its purification. For this reason,no innovations have been made to add additional functions to the designof bioreactors while they do provide a large investment in a vessel thatcould possibly have multiple uses.

There is a large unmet need to stream line the entire process ofbiological manufacturing of products where the cost of manufacturing canbe reduced substantially by combining several traditional steps in asingle vessel, the bioreactor.

The instant invention discloses an innovative bioreactor design thataccomplishes this goal and is applicable universally to all types ofbioreaction applications.

SUMMARY OF THE INVENTION

There are two major types of recombinant expressions of biologicalproducts. One is the soluble form of biological product that is secretedinto nutrient medium by the cells as most often seen in the use ofChinese Hamster Ovary cells and the other is the retention of biologicalproduct inside the cell forming an inclusion body, as most often seen inthe case of using E. Coli for expression. Recent advances in geneticengineering have been able to encode the genes of bacteria that wouldsecrete soluble proteins instead of retain them inside as inclusionbodies. This is to avoid the cumbersome process of cell-lysis andinclusion body solubilization.

Historically, biological products expressed in nutrient medium areseparated form the medium by first removing the biological culture by aprocess of centrifugation or filtration. This step is followed byreducing the volume of medium to about 1/10 to 1/20 to make it possibleto load the liquid within a reasonable time on purification columns.While these process steps have been widely validated and function verywell, the practicality of using these steps becomes very difficult whenlarge volumes of medium is handled.

Today, it is not uncommon to see bioreactors processing thousands andeven hundreds of thousands of liters of medium at a time. To accommodatethis, companies use very large-scale filtration and volume reductionmethods that cost millions of dollars to install and millions more tooperate and maintain. There is a very large unmet need to simplify theseprocesses, reduce the cost of production and make the technologyaccessible to thousands of researchers and smaller companies who cannotafford such large investments. Circumventing these process hurdles wouldalso make it possible to produce drugs based on these biological drugscheaper to manufacture and thus increase their affordability to billionsof people around the world who are not able to afford these drugs.

The key to the instant invention lies in following a contrarianteaching. While all manufacturers follow the path described aboveinvolving removal of components from a broth ready for purification, itwould be prudent to examine the utility of first removing the targetbiological product instead and discarding what is not needed, instead ofremoving step by step what is not needed, as currently practiced.

The instant invention capitalizes on the recent availability of manyresins that are capable of binding biological products in largequantities. Most modern resins would bind between 20-125 mg ofbiological product per mL of resin. Many of these resins are highlyspecific to the biological products and many of them can be combined toremove any type and quantity of a biological product from a solution bya simple process of physicochemical binding that is strong enough toretain the biological products attached to the resin while the culturemedium is removed from the bioreactor. The art has also advancedsignificantly in the field of biological product purification wherein wenow have a much better ability to elute these bound biological productsfrom resins by adjusting the pH, the ionic strength or othercharacteristics of the eluting buffer to break the binding between theresin and the biological product. This allows removal of biologicalproducts from a bioreactor as a highly concentrated solution that isready for further purification and in some instances it can even be thefinal product for use.

The bioreactor design of the instant invention is novel, and overcomesthe most significant hurdles in the namufacture of biological productsby applying a contrarian teaching in the current method of themanufacture of these products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a flexible two-dimensional disposable bioreactordisplaying the installation of the pouch used for adding resin to thebioreactor to separate the biological products form the bioreactor.

DETAILED DESCRIPTION OF INVENTION

Affinity Chromatography is a separation techinque based upon molecularconformation, which frequently utilizes application specific resins.These resins have ligands attached to their surfaces, which are specificfor the compounds to be separated. Most frequently, these ligandsfunction in a fashion similar to that of antibody-antigen interactions.This “lock and key” fit between the ligand and its target compound makesit highly specific.

Many membrane proteins are glycobiological products and can be purifiedby lectin affinity chromatography. Detergent-solubilized proteins can beallowed to bind to a chromatography resin that has been modified to havea covalently attached lectin.

Immunoaffinity chromatography resin employs the specific binding of anantibody to the target biological product to selectively purify thebiological product. The procedure involves immobilizing an antibody to acolumn material, which then selectively binds the biological product,while everything else flow through.

Inclusion bodies upon solubilization exposes hydrophobic groups whilethere remain chemical groups on denatured proteins capable of binding toresin (singh and Panda, 2005), allowed separation of these proteinsduring the stages of refolding to native state.

Some of the state of the art resins binding technologies include:

-   -   Novozymes's newly patented Dual Affinity Polypeptide technology        platform replaces Protein A process steps with similar, but        disposable, technology.    -   Stimuli responsive polymers enable complexation and manipulation        of biological products and allow for control of polymer and        biological product complex solubility, which results in the        direct capture of the product without centrifuges of Protein A        media, from Millipore Corp.    -   Mixed mode sorbents to replace traditional Protein A and ion        exchange, for improved selectivity and capacity with shorter        residence times. These media, with novel chemistries, include        hydrophobic charge induction chromatography, such as MEP, and Q        and S HyperCel from Pall Corp.    -   Monoliths, involving chromatography medium as a single-piece        homogeneous column, such as Convective Interaction Media        monolithic columns from BIA Separations.    -   Simulated moving beds, involving multicolumn countercurrent        chromatography, such as BioSMB from Tarpon Biosystems.    -   Protein G (multiple vendors).    -   Single domain camel-derived (camelid) antibodies to IgG, such as        CaptureSelect from BAC.    -   New inorganic ligands, including synthetic dyes, such as        Mabsorbent A1P and A2P from Prometic Biosciences.    -   Expanded bed adsorption chromatography systems, such as the        Rhobust platform from Upfront Chromatography.    -   Ultra-durable zirconia oxide-bound affinity ligand        chromatography media from ZirChrom Separations.    -   Fe-receptor mimetic ligand from Tecnoge.    -   ADSEPT (ADvanced SEParation Technology) from Nysa Membrane        Technologies.    -   Membrane affinity purification system from PurePharm        Technologies.    -   Custom-designed peptidic ligands for affinity chromatography        from Prometic Biosciences, Dyax, and others.    -   Protein A- and G-coated magnetic beads, such as from        Invitrogen/Dynal.    -   New affinity purification methods based on expression of        biological products of MAbs as fusion biological products with        removable portion (tag) having affinity for chromatography        media, such as histidine) tages licensed by Roche (Genentech).    -   Protein A alternatives in development, including reverse        micelles (liposomes), liquid-liquid extraction systems,        crystallization, immobilized metal affinity chromatography, and        novel membrane chromatography systems.    -   Plug-and-play solutions with disposable components (e.g.,        ReadyToProcess), process development ÄKTA with design of        experiments capability, and multicolumn continuous capture, from        GE Healthcare.

It is surprising that while great advances have been made in the designof resins available to capture biological products, these have been onlyused in the downstream processing of purification. Adding resins to acrude mixture of biological products and host cells would be nodifferent than the current process that simply concentrates the samemedium and loads it onto columns. The only difference would be that whenpracticed at the end of the bioreaction cycle, this would requiresufficient resin to bind almost the entire biological product. Assumingthat a cell line produces G/L of protein and the binding capacity is 0.1G/mL, this will require 10 L of resin when operating a 1000 Lbioreactor. When operating the instant invention to replace a perfusionsystem or continuously remove the biological product, smaller quantitiesof resins are required as they can be removed from the bioreactor andreused. However, if we examine the lifecycle of resins, the total costof resin use would still be the same. With the cost of resins droppingsubstantially and the possibility of using non-specific binding resinsreduces the cost burden of processing the entire yield at one time. Thecost savings in the instant invention comes in the reduced time ofapproximately 50% in processing, allowing manufacturers to prepare alarger number of batches reducing the cost or production. Additionally,one embodiment of the invention may be practiced by using the leastexpensive resins to generically bind all soluble organic components andthen elute them instantly using a buffer without any concern for theprofile of elution to separate these components. Such generic resins arevery inexpensive and may not even have to be reused.

The instant invention offers four methods of biological productmanufacturing and purification. The first method is the separation of abiological product at the end of the bioreaction cycle; the secondmethod is the removal of biological product continuously while the resinremains inside the bioreactor and the third method involved periodicallyremoving the resin and processing it outside the bioreactor. In thefourth method, the cells are lysed to expose inclusion bodies, which arethen solubilized prior to contacting them with resin.

The first method would be the most commonly practiced art; the secondmethod would be needed to obviate the toxic effects of expressedbiological products and to replace a perfusion bioreaction method andthe third method would be practiced when the biological product may notbe stable in the complex stage or where it is desired to re-use theresin (particularly where the cost of the resin is high). The fourthmethod would be useful for bacterial expressions that involve formationof inclusion bodies and while newer constructs allow expression ofsoluble products, there remain a large number of existing methods thatinevitably require handing inclusion bodies.

One of the main objectives of the instant invention is to eliminatecertain unit processes, which are cumbersome and expensive. The instantinvention introduces a concept of filtering out biological culture usedin the bioreaction. Most bacteria would be about 8μ in diameter and theChinese Hamster Ovary cells about 5μ in diameter. The binding resin isseparated from the culture broth by a container device (pouch) that isporous and the porosity of the walls of the container is kept at below5μ to prevent entry of any organism or cell into cnad contacting theresin. This prevents any physical adsorption of cells or organisms onthe resin and fouling it. Additionally, during the drain cycle, when theliquid content of the bioreactor is drained, this removes all cells andorganisms. Of course, any components of lysed cells or organisms wouldindeed enter the pouch and contact with the resin and will be retainedby the resin. But this too is a process that is common with the presentpractice. In some instances, it may be necessary to use the pouches withlarger diameter of 30μ or higher and this may cause some deposition ofcells on the resin. For such instances, the invention adds an optionalstep of filtering the buffer eluted through a sterilizing filter toremove any remaining cells that might elute along with the biologicalproduct and those that may have remained attached to the resin duringthe wash cycle. Buth this stage of filtration will be much lesscumbersome as smaller volumes are filtered with essentially 95% or morereduction in the filtration load and the risk of clogging of the filtersince most of the culture has already been drained. For example, insteadof filtering a 2000 L culture media to remove CHO cells, this willrequire filtering about 100 L or less; the cost and time savings wouldstill be high.

It is noteworthy that downstream processes are required to remove hostcell and DNA biological products effectively. Current methods start witha substantial load of these biological products despite the filtrationof the cells, so the instant invention does not add any new burden onthe complexity of downstream processing.

Another most significant advantage of the methods disclosed comes inincreased production yields. It is well established that the process offiltration, which is conducted under high pressure, inevitablydecomposes biological products. By avoiding the filtration steps, it isenvisioned that the product yields will improve substantially.

The very nature of the recombinant product makes it unstable. Theinstability of a recombinant biological product can be either physicalor chemical. Physical instability can be related to such things asdenaturation of the secondary and tertiary structure of the biologicalporduct, adsorption of the biological product onto interfaces orexcipients, and aggregation and precipitation of the biological product.Chemical instability of a biological product results in the formation ofa new chemical entity by cleavage or by new bond formation. Examples ofthis type of instability would be deamidation, proteolysis andreacmization. Any changes to the manufacturing process that reduce thecycle of production, exposure to harsh conditions such as high pressuresacross membranes in cross-flow and sterile filtration, etc., wouldincrease the stability and the fianl yield of production.

In one method of batch processing, the biological products are harvestedat the end of the cycle that might be as long as several weeks ofcontinuous expression. While many biological products would survive the37° C. environment for that length of time, a few would degrade overthat period of time. By capturing the biological products throughformation of resin-biological product complex, the stability of and thusthe yield of production can be increased since in the complex stage, themolecules are immobilized and thus less likely to degrade. While manybiological products may degrade by adsorbing to various surfaces, theinteraction between a resin and biological product is of a differentnature as evidenced by the use of resins in the purification ofbiological products whereby high degree of stability is maintained wheneluting from a resin column.

Another significant advantage of the methods disclosed here occurs inreducing the toxicity of the biological products expressed to the celllines expressing them. The resin may be placed inside the pouch from thevery beginning of the reaction process and as biological product isexpressed, it is instantly captured by the resin removing from directcontact with the cell lines increasing their productivity and thelongevity of expression cycle, decreasing the production costssubstantially.

In a biological system, a particular biological product is expressedonly in a specific subcellular location, tissue or cell type, during adefined time period, and at a particular quantity level. This is thespatial, temporal, and quantitative expression. Recombinant biologicalproduct expression often introduces a foreign biological product in ahost cell and expresses the biological product at levels significantlyhigher than the physiological level of the biological product in itsnative host and at the time the biological product is not needed. Theover-expressed recombinant biological product will perform certainfunction in the host cell if the biological product is expressed solubleand functional. The function of the expressed recombinant biologicalproduct is often net needed by the host cell. In fact the function ofthe biological product may be detrimental to the proliferation anddifferentiation of the host cell. The observed phenotypes of the hostcells are slow growth rate and low ecll density. In some cases, therecombinant biological product causes death of the host cell. Thesephenomena are described as biological product toxicity. Theserecombinant biological products are called toxic biological products.

Biological product toxicity is a commonly observed phenomenon. Allactive biological products will perform certain functions. The hostcells need all of these functions with few exceptions and therefore,they interfere with cellular proliferation and fifferentiation. Theappeared phenotype of the effects of these biological products to thehost cells is their “toxicity.” It is estimated that about 80% of allsoluble biological products have certain degree of toxicity to theirhosts. About 10% of all biological products are highly toxic to hostcells. The completely insoluble or dysfunctional biological productswill not be toxic to the host cell, though they may drain the cellularenergy to produce them when over-expressed. Biological productover-expression creates metabolic burden for the host cell, but thisburden is not toxicity to the cell. Some low solubility or partiallyfunctional biological products may still be toxic to the host. While theexposure of the host cell to biological product being expressed isinevitable and is only optimized through codon usage, once thebiological product has been expressed, it would be prudent to transportit out of the cell as soon as possible and this diffusion reactionrequires establishing a sink condition that is readily achieved if theexpressed biological product in the surroundings of the host cell isremoved from the solution such as in the case of the instant inventionby binding to a resin.

In another situation, where a perfusion system is used for the upstreamproduction of recombinant biological products, a portion of culturemedia is replaced with fresh media and the media removed is filtered ofhost cells, reduced in volume and wither stored at a lower temperatureor processed with downstream processing. Still another significantadvantage of the method disclosed, comes in performing a perfusionbioreaction. The traditional process of perfusion can be replaced bysimply removing the biological product from the solution by adding aresin to the pouch and replenishing any nutrients that may have beenlost due to adsorption onto the resin. There is a substantial costreduction in using this substitute method.

It is noteworthy that the instant invention allows for provisions tokeep the bioreaction going at its optimal conditions by replenishing anynutrients lost to the binding resin. This may happen when the resinsused have non-specific binding characteristics. Where highly specificaffinity binding resins are used, this step may be obviated or reducedin its frequency.

And yet another advantage of the disclosed method is that the finalresin-biological product conjugate can be loaded directly column andeluted accordingly to specified protocols without firts flushing it outwith a buffer to break the bonding between the resin and the biologicalproduct. This will save substantial time and material savings.

A remarkable application of the instant invention is made in themanufacturing of recombinant biological products using bacterialculture. E. coli has been most widely used for the production ofrecombinant proteins that do not require posttranslational modificationssuch as glycosylation for bioactivity. A typical process involves,harvesting bacteria by a process of centrifugation, to collect the cellpaste. Since the high-level expression of recombinant proteins resultsin accumulation of protein as insoluble aggregates as inclusion bodies,the cells are lysed, most commonly by a sonication process and theinclusion bodies solubilized (by the use of a high concentration ofdenaturants such as urea or guanidine hydrochloride, along with reducingagents such as beta-mercaptoethanol), refolded (by slow removal of thedenaturant in the presence of oxidizing agent) and purified to recoverfunctionality of the active product. Protein solubilization from theinclusion body using high concentration of chaotropic reagents resultsin the loss of secondary structure leading to the random coil formationof the protein structure and exposure of hydrophobic surface, a featurethat is of significant inportance in the instant invention.

One embodiment of the instant invention combines several procedures ofcell lysis, solubilization and refolding into one continuous operationthat can all be completed within the bioreactor, obviating the need formultiple vessels, handing large volumes of liquids and reducing processtime and cost of manufacturing.

Protein production in Escherichia coli involves high-level expression ina culture, followed by harvesting of the cells and finally theirdiruption, or lysis, to release the expressed proteins. One of the mostcrucial steps to be optimized in the protein production process isbacterial cell lysis. Although bacterial cell lysis does not influenceprotein expression, it can have an effect on protein solubility byaffecting the physicochemical properties of the protein. chemical lysiscan be achieved by using different buffer composition, lysozyme, orcommercially available detergent reagents. Cell lysis can also include acombination of the mechanical and chemical lysis, e.g., lysozyme withfreeze-thaw cycles. The preferred method, or “gold standard”, forbacterial lysis on the small or standard laboratory scale production issonication. It relies on the mechanical disruption of the bacterial cellwall. Any solubilizing lysis agents, like detergents, that can affectsolubility or stability, do not affect the expressed protein. Sonicationbecomes more problematic when handling large volumes of culture media.For these reasons, many high throughput laboratories choose to optimizelysis conditions by chemical means.

Chemical lysis includes the treatment of cells with alkali, enzyme, ordetergents. Chemical lysis methods minimize denaturation and expose theinner, cytoplastmic membrane by degrading the peptidoglycan cell wall ofbacteria. The cell wall of Gram-positive bacteria is thick, containingseveral interconnecting layers of peptidoglycan (60-90% of the cellwall). In contrast, the cell wall of Gram-negative bacteria appearsthin, containing two or three layers of peptigoglycan (10-20%) of thecell wall). In addition to this, Gram-negative bacteria contain an outermembrane composed of lipopolysaccharide, phospholipids, and lipoprotein.Lysozyme, a commercial lytic enzyme, is widely used to lyseGram-positive cells in the presence of EDTA and detergent Brij 58.Lysozyme hydrolyzes N-acetylmuramide linkages, resulting in degradationof bacterial cell walls. The activity of lysozyme is optimal in the pHrange of 6.7 to 8.6.

In contrast, gram-negative bacteria are less susceptible to lysozyme anddetergents due to the presence of asymmetric lipid bilayer. The outermembrane of the peptidoglycan acts as a permeability barrier to largemolecules, and so the outer membrane needs to be permeabilized to exposethe peptidoglycan layer for successful enzymatic lysis. The permeabilitybarrier is, in part, due to the presence of polyanioniclipopolysaccharide that provide a network interaction in the presence ofdivalent cations, such as Mg2+. The chelators of divalent carions (e.g.,EDTA), polycationic species, and small molecules (e.g., Tris) aresuitable for permeabilizing the membrane in order to releaselipopolysachccharides.

Chemical cell lysis can be performed using lysis solution containingeither lysozyme (Sigma-Aldrich, St. Louis, Mo.). SoluLyse® in Trisbuffer (Genlantis, San Diego, Calif.) or Bugbuster® protein extractionreagent (Novagen, EMD Chemicals Inc., San Diego, Calif.) The amount ofsoluble protein and the percentage recovered in the soluble fractionusing SoluLyse® well correlates with sonication. Compositions andprotocols for chemical lysis are widely available through commercialsuppliers of chemical lysis products. The quantity of various chemicalsused, the time of exposure and determination of the end point arereadily established for any specific process.

Solubilizing the lysed cell product would yield a denatured protein withlarge hydrophobic and ionic surfaces that can be readily bound to resinslike cationic, anionic or hydrophobic resins; in some instances, certainsolution characteristics like the pH, ionic strength of polarity mayhave to be adjusted to achieve optimal binding to the resin introducedin the pouch. This will allow discarding of the large volume of liquidculture medium and cell debris; it is noteworthy that the pore size ofthe pouch would generally be small enough to exclude cell lysis debristo contact the resin.

The solubilized proteins bound to resins can then be removed from thebinding and a solution of protein allowed to refold inside thebioreactor and again once the refolding has been completed, binding theproteins to resin and discarding the refolding solution obviating theneed for expensive and time consuming cross-flow filtration operations.The concentrated solutions of refolded proteins are then subjected tofurther purification.

There remains a large unmet need to develop a technology wherein thetarget biological product is selectively or non-selectively removed fromthe culture media prior to subjecting it to customary purificationprocesses. The instant invention, taking a contrarian approach, istargeted to modify the existing designs of bioreactors to include a stepof performing biological product harvesting or biological productcapturing prior to purification chromatography steps to increase thethroughput of manufacturing processing without adding expensive andtechnically challenging modifications.

The key component of the instant invention lies in a feature added to atraditional bioreactor, whether a hard-walled system or a flexibledisposable system. A pouch made of a porous material (likely a polymericor metallic mesh) with porosity that is smaller than the size of resinused to capture biological products is used to allow contacting of theresin with the biological product. Most resins come in sizes rangingfrom 50 microns and up; some have smaller particle size as well.However, it is possible to design a pouch, a bag or a container form apolymeric material such as nylon that would keep the resin within thebag and not allow it to enter to the culture medium when the pouch isplaced inside a bioreactor. To make sure that smaller particles of theresin are not flushed out of the bag carrying biological products withthem, it would be necessary to sort out the resin first by placing it ina similar bag as installed in a bioreactor and immersing it in water toflush out any smaller particles. It is noteworthy that the resins,though expensive, can be re-used numberous times without losing theirefficacy of binding and even when they do, the method described hereallows for adjusting the quantity of resin to achieve maximum capture ofbiological products.

A significant advancement in the art of biological product capture isprovided here by disclosing that a mixture of resins can be used toobviate the binding of sites on the resin by other functional groupsfound in the culture media. The ultimate goal is to design a mixture ofresins that would always capture the all of the biological products inthe culture medium within the shortest period of time. Once used, theresins can be cleansed, sanitized and readied for the next use. It isimportant to know that there is no need for sterilizing these resins aslong as they are treated chemically to reduce the microbial load.

Recent advances in the sensors available for bioreactors now make itpossible to monitor many properties including dissolved oxygen,dissolved carbon dioxide, electrolyte conecntration, pH, turbidity, cellcount, temperature, and also the concentration of dissolved biologicalproducts, all by using non-invasive methods. The instant invention canbe automated by installing such sensors and more particularly a sensorto determine concentration of the biological product so that the resincan be added to the pouch at a certain time when the concentration ofthe biological product in culture media has reached a pre-determinedhigh level and allowing it to equilibrate until such time that theconcentration in the culture media decreases to a certain pre-determinedlow level, most likely below 1% of the highest level prior to thetreatment with resin.

Common Embodiments

In a first embodiment, the instant invention proposes a bioreactorcapable of growing all types of cells and organisms and additionallyprovides a ready means of harvesting of biological products in abioreactor. The instant invention employs a mechanical device, which inone step combines several steps or biological product harvesting. Themethod of the present invention presents a novel procedural step forsimultaneously extracting and concentrating a biological product ofinterest from a host cell, at the same time removing practically all, orat least the majority, of the host cell biological products.

In a second embodiment, the present invention relates to a bioreactorthat contains a resin capable of binding target biological products butkept separate from the culture medium by placing it inside a pouch thathas porous walls with pores small enough to hold the resin inside thepouch yet allow the culture media containing target biological productto freely equilibrate with the resin. By placing the resin in a pouch,several arduous steps in protein harvesting are avoided. It is thepurpose of this invention to work the purification process in anopposite order to how the art is currently practiced universally. In allinstances, upon the completion of the bioreaction, the dirst step is toremove the host cells or organisms by filtration or centrifugation. Withthe bioreactor volumes into thousands of liters, this process isextremely arduous, expensive and requires additional storage vessels ofabout the same size as the bioreactor making it difficult to accommodatethese processis in smaller facilities. A goal of this invention is toreverse the process and instead of removing the host cells andorganisms, remove the biological product first. This modification alsoeliminates the need to reduce the volume of filtrate received afterremoving the host cells and organisms in the traditional process priorto purification. To accomplish this, the bioreactor contains a pouchthat is filled with a resin when the process is ready for harvesting,allowing equilibration of the binding process and the draining out theculture media along with host cells and organisms.

The drainage is best accomplished by allowing the culture media to flowdown under gravity, thus obviating any steps that might take a long timelike peristaltic pumping of the culture medium out of the bioreactors.The biological product is eluted using a buffer that causes breakdown ofthe association between the biological product and the resin andcollecting a concentrated solution. Prior to contacting theresin-biological product complex with a buffer, the complex can bewashed if necessary with fluids that would not break down theresin-biological product complex but remove other components bound toresin that may have come from the metabolic products in the culturemedia. Just in case there are any host cells remaining, this solution,which would be about 2-5% of the volume of the culture media, can beeasily filtered through a sterilizing filter.

In a third embodiment, the instant invention obviates the need forcostly cross-flow filtration processes used in every type ofmanufacturing of biological products as in almost all instances aconcentration step is involved to reduce the volume of liquid that isloaded onto purification column. The purification of biologicaltherapeutics generally involves the use of cross flow filtration(tangential flow filtration), normal flow filtration (dead endedfiltration) combined with chromatographic separations. Cross flowfiltration and normal flow filtration retain matter through sizeexclusion and are complementary to chromatography's selectivity. Forprocesses where volumes are large such as into thousands of liters, thecost of equipment for filtration is into hundreds of thousands ofdollars with expensive filters all adding to a cost that represents amajor fraction of the total cost of manufacturing of recombinant drugs.

In a fourth embodiment, the instant invention provides a means ofcontinuously removing expressed biological product from a culture mediato enhance the level of expression that may be depressed because of thehigher concentration of biological product in the mixture. The instantinvention allows maintenance of a sink condition for the concentrationof the biological product at all times.

In a fifth embodiment, the instant invention provides a means ofcontinuously removing expressed biological product from a culture mediato reduce the toxicity of the expressed biological product to host cellsand thus prologning the cycles of expression substantially increasingthe yields of production.

In a sixth embodiment, the instant invention provides a means ofincreasing the chemical stability of expressed biological product bybinding it to a resin as soon as it is expressed as the chemicals arealways less stable in a solution form than in a solid form or in thiscase a complex form. this would substantially improve the yield ofproduction.

In a seventh embodiment, the instant invention provides a means ofsubstantially reducing the cost of recombinant drug manufacturing byeliminating some of the most costly and time consuming steps. The costof using a non-specific resin is minimal as this can be used repeatedly,unlike the resin used in the downstream purification where it must bereplaced periodically as it loses its power to resolve the separation.Until the resin reaks down or is physically damaged, it can be usedcontinuously and even when the efficiency of adsorption is reduced, itcan be mixed with fresh resin to give it a very long useful life.

In an eight embodiment, the instant invention combines several steps ofupstream and downstream bioprocessing. In the harvesting process, theresin-biological product complex can be directly treated with buffers tobegin the first stage of purification and where the resin is carefullyand artfully selected, lead to high purity of a biological product inone step. The resin-biological product complex is ready for downstreamprocessing without the need to load a column intended for downstreamprocessing and this can save substantial time for loading. The prolongeddelay in loading columns as currently practiced is often detrimental tothe stability of target biological product. This can be avoided usingthe instant invention.

In a ninth embodiment, the instant invention offers to eliminate a verylaborious and expensive step of first stage filtration or other means ofseparating the biological product harvested. By using a pouch to containthe resin, all steps generally required to remove resin such asdecanting, centrifugation (low speed), filtration (coarse) can beavoided altogether. The pouches can be stringed together so that theseare simply removed by picking up the end of the string at one end. Thepouches can also, then, be packed directly in a column for elution as ifthis were loose resin. Since the pouches containing the resin can bepre-washed to remove the resin of particle size smaller than theporosity of the filter that forms the pouch, the losses of boundbiological product to resin will be eliminated. The pouches can bewashed and re-used, perhaps requiring a sterilization step if these areused during the bioreaction cycle, a chemical can achieve thesterilization similar to what is used in the sanitization of thechromatography column. This method of holding the resin in a pouchfurther reduces any loss of resin and saves additional costs.

In a tenth embodiment, the instant invention describes a novel method ofbiological product purification wherein all those steps which areexpensive and time consuming are obviated; the method of biologicalproduct purification involves adding to a solution of biological productready for purification, a resin contained in a pouch that is the firstresin to be used in the process of purification. Once the biologicalproduct binds completely to the resin, the resin is packed into apurification column. This method of loading the biological product in apurification column is more efficient than the traditional method ofcalculating the capacity of binding of resin and thus determining thevolume of resin used. There are always possibilities of miscalculationssince the binding of the biological product to the resin is dependent onmany factors, e.g., the physicochemical characteristics of the liquidloaded. These characteristics would vary in every batch; thecalculations of the amount of resin required are at best goodtheoretical guesses. Loading too much protein would cause loss ofprotein and adding too little, add to the cost of resin. The instantinvention allows for a perfect match of the binding capacity to thequantity of the biological product bound, as it is possible to monitorthe concentration of the unbound concentration of the biologicalproduct. Once the quantity of resin used is such that the concentrationof the biological product in the nutrient medium is reduced to apre-determined level, it is assumed that all protein has been bound.

In an eleventh embodiment, the instant invention provides a method ofextraction of solubilized inclusion bodies by lysing the cells in thebioreactor, solubilizing the inclusion bodies and capturing them with aresin to remove them from the bioreactor. This application substantiallyreduces the cost of manufacturing of proteins, which are expressed asinclusion bodies.

The overall impact of these embodiments is quantifiable in terms of thetime it takes to make a biological product ready for purification; as ageneral guideline, if a 2000 L batch of a recombinant production isready for processing, it will take about 10-12 hours to filter itthrough a 0.22μ micron filter to remove host cells such as ChineseHamster Ovary Cells. This step would then be followed by a cross-flowfiltration process that might take 12-24 hours to reduce the volume to200-300 liters. This step is then followed by loading on the column,which may take another 6-24 hours depending on the size of the columnused. While the batch is subjected to the above processes, the targetbiological product is undergoing degradation, both because of theeffects of temperature as well as the strain exerted on biologicalproducts in the filtration process. The instant invention offers asolution to replace all of these steps with a single short step with atime savings of at least 50% in the overall process time and materialsavings of about 30% and improved yields of about 20%.

Bioreactor Design

FIG. 1

FIG. 1 shows a preferred embodiment of the invention. The bioreactorconsists of a two dimensional disposable flexible bag (1) resting on asupport surface (10), which is capable of being tilted and furtherresting on a frame (9), a means of gassing consisting of a gas inlet(2), a gas filter (3) and a sparging tube (4). The flexible bag (1)further contains a gas outlet (5), a nutrient medium inlet (6), a liquiddrain (14) controllable by a stopcock (13), a means of heating (8) thesupport surface (10) a means of agitating the nutrient medium comprisinga flapper (7) that compresses on the flexible bag (1) intermittently;also provided in the preferred embodiment of the invention is a pouch(12) with a resin inlet/outlet (11) to add or remove the resin.

The above-preferred embodiment of a bioreactor design would be useful inthe manufacture of all types of biological products using all types ofcells and organisms. The bioreactor is operated by first adding a fixedvolume of a nutrient medium to the flexible bag, which would generallybe supplied, pre-sterilized by gamma radiation. The nutrient medium maybe sterile filtered directly into the bag for convenience. The bag wouldrest on a supportive surface that can be tilted if needed. Generally,for bag sizes of up to 36 inches, this may not be necessary; otherwisethe support surface can be raised on one side by an angle of 0.1 to 5degrees.

This slight tilt of the supportive surface adds potential energy to thenutrient medium and causes it to draw more towards the flapper (7). Theflapper mechanism is turned on resulting in the flapper compressing onthe flexile bag at one end of the bag. This compression produces a waveinside the bag that travels to the other end of the bag and thenresturns after stricking the other end of the bag; a slight tilt, ifutilized, assures that there is no accumulation of unmixed media at theother end of the bag opposite to the end of bag being compressedperiodically. The flapper would generally be operated at a rate of 25-60rpm depending on the volume of nutrient medium, and the size of the bagused. Generally, the flexible bag would be filled to about 60% of thecapacity. Once the flapper mechanism has begun operating and the mixingseems adequate as evidenced by smooth moving of nutrient medium insidethe bag, the heating element (8) is turned on to achieve a desiretemperature inside the bag. Sensors may be attached to the bag to recordthe temperature and connect these sensors to a feedback heatingmechanism that would assure maintenance of an appropriate temperaturesuch as 37 C. These sensors are not shown in FIG. 1 as they arecustomary and generically available. Alternately, a sample of nutrientmedium may be drawn to measure its characteristics. Once the temperaturereaches the desired level, a biological culture of a recombinantorganism such as Chinese Hamster Ovary cell or E. coli would be added tothe nutrient medium through the nutrient medium inlet and the bagallowed mixing. Alternately, the biological culture may be added at anytime, even before adding the nutrient medium. The gas is turned on tobegin sparging of the nutrient medium at a rate predetermined to besuitable for the specific process.

For an E. Coli expression experiment, the flow rate would beapproximately 0.8 to 1 vvm of compressed air. The key to achieving bestaeration and the highest KLA value is to allow the bag to inflate onlyslightly, to allow sufficient surface for the sparged air to escape, yetnot cause pressurization of the bag. It is for this reason that the airoutlet is carefully controlled for the outlet rate. Once a steady stateof flow rate, bag pressurization and mixing dynamics is achieved, thebioreactor is allowed to run, such as overnight when using for bacterialfermentation or for several days when using Chinese Hamster Ovary cells.During this period, the nutrient medium may be fed with nutrientsthrough the media inlet tube. The optical density of bacterial cultureor the cell density, dissolved oxygen and pH can be carefully monitoredto assure the optimal condition for the expression of biologicalproducts in the nutrient medium. While the preferred embodiment wouldfunction only when the biological product is present in a solution formin nutrient medium, the biological processes that produce an inclusionbody can also benefit from the instant invention if the cells arechemically lysed and the inclusion bodies solubilized.

It is now well established in prior art that solubilized inclusionbodies can be loaded onto resin columns to perform refolding of proteinsand thus there exist a large number of resins that would quickly andefficiently bind solubilized inclusion bodies. Thus the instantinvention is applicable to bacterial production even if they do notdirectly express soluble proteins. However, the process of manufactureof the biological products would invelve a chemical treatment to lysethe cells and then chemically solubilize the inclusion bodies prior tomoving to the resin-binding step.

The next step is to calculate the amount of resin needed to bind thebiological product based on the concentration of the biological productin the nutrient medium. The resin is first prepared by removing resinparticles that would be smaller than the pore size of the pouch (whichwould generally be about pb 3μ). The sized resin is then introduceddirectly into the pouch through the pouch inlet and the nutrient mediumallowed to agitate while the gassing is turned off. [It is expected thatmore than 99.9% of all resins used would have particle size larger than3μ and thus no bleeding of resin will take place back into bioreactor}.Samples of nutrient medium are taken periodically to ascertain when themajority of the biological product has become bound to the resin insidethe pouch. Generally, this would be above 90% reduction in theconcentration of the biological product in the nutrient medium. It maybe necessary to add more resin if the concentration of the biologicalproduct does not reach a pre-determined low level within apre-determined time. The time needed for such equilibration will beabout 20-30 minutes however, specific binding rate studies would need tobe conducted to assure that an optimal minimal time is allowed for suchequilibration.

Once it is determined that an optimal binding of the biological producthas been achieved, the drain port is operated by opening the stopcockand the culture media along with cells or organisms is allowed to flowout under gravity force. For large-scale operations, it may be necessaryto install the drain port towards one end of the bag and the bag tiltedto force fast removal of the nutrient medium from the bag. Theutilization of gravity flow is a major energy and time saving feature.In those instances where thousands of liters of nutrient media is used,any mechanical process for moving or handling nutrient media would be aninefficient process compared to gravity flow resulting in discarding ofnutrient media. It is further emphasized that the NIH guidelines forLSGP (large scale good practice) allow many recombinant cells to bedirectly discarded into sewer without any treatment as they are notinfectious. This is particularly true of the Chinese Hamster Ovary Cellsthat comprise the largest production engine in bioprocessing. Even someE. Coli bacteria are exempted from any decontamination step. In suchsituations, the most energy and cost efficient process is draining ofnutrient media directly into sewer. The size of the drain would have tomatch with the flow rate desired and where large volumes are used,several drains can be installed in the bioreactor to quickly andefficiently remove nutrient medium. Once the nutrient medium has beendrained out, the stopcock in the drain is closed. At this stage, themanufacturer would have two options, one is to fill the bag with abuffer that would not cause the breaking of the binding between thebiological product and the resin but would be generally effective inremoving other smaller molecular weight components that might havebecome attached to the resin. The bag would then be agitated for a briefperiod of time and the buffer (which may even be water) drained outagain by turning on the stopcock in the drain port. This would be thewashing step. This step can be skipped and the bag filled with a buffersolution that would cause the breakdwon of the binding between thebiological product and the resin. This would generally require a pHadjustment, a polarity adjustment and an electrolyte adjustment.

These conditions would have already been worked out in the early phasesof process development. Once the breaking or eluting buffer is allowedto react within the bag, the biological product would be released intothe buffer solution, which can be monitored for the concentration of thebiological product to assure that a desirable recovery has beenachieved. While the goal is to recover almost the entire biologicalproduct, it may at times be more useful to settle with a more practicallevel of recovery such as 90 to 95%. Once this stage has reachedm,turning on the stopcock again opens the drain port and the concentratedsolution of the biological product is collected in a microbiologicallyclean vessel. Generally, the volume of the solution will be about 2-5%of the original nutrient medium. This concentrated solution would thenbe transferred to downstream purification columns. In most instances, itmay be desirable to pass this concentrated solution through asterilizing filter to remove any cells that might have been carried overto prevent the blocking of the purification columns. It is expected thatthe titer of cells at this stage will be very small allowing use ofsimpler and faster filtration methods and even if the solution is notfiltered, the chance of blocking the purification column would beminimal. Smart manufacturing processes have the fewest steps involved.The manufacturers would be advised to consider eliminating thisfiltration step, if possible.

In another preferred embodiment, the bioreactor is operated as providedin the embodiment above, except that the resin is added in the beginningof the bioreaction cycle to bind the biological product as it isexpressed in the nutrient medium. This technique would then not beapplicable to situations where the biological product is expressed asinclusion bodies. Smaller portions of resins are added periodically tothe pouch in quantities just enough to bind the expressed biologicalproduct. This is necessary to prevent blockage of binding sites on theresin by other adsorbable materials in the nutrient medium, and tominimize the losses of nutrient elements to the resin, which should bereplenished periodically. This method would be analogous to atraditional perfusion system except that the steps to replace the media,filtering it and replacing media with fresh media are obviated. Thismethod is also a useful method to reduce the toxicity of the biologicalproduct to the host cells expressing the biological product. This methodwould work well for those biological products that can stay stab le whenbound to resin as the complex between the biological product and theresin is kept in the pouch until the end of the bioreaction cycle.

In another preferred embodiment, a further improvement is made in thepreceding embodiment where the resin is added periodically. In thispractice of the method, the resin is first removed that has becomeequilibrated with the biological product prior to adding fresh resin.This allows the removal of biological products bound to resin and avoidsany instability problems due to keeping the conjugate of biologicalproduct and resin in the bioreactor for a prolonged time. The resin isremoved readily by inserting a tube from the inlet/outlet to the pouch,which would generally be of a cylindrical shape and aspirating theresin, using vacuum. It is noteworthy that the size of the tube insertedis such that it fits snugly inside the tubular (cylindrical) pouchpushing the resin as the tube goes down and forcing the resin into thetube by mechanical displacement. Once the tube has settled down deepinto the pouch, a vacuum is applied carefully avoiding aspirating anysubstantial quantities of the nutrient medium. The resin collectedperiodically can be kept at a more suitable temperature and combined atan appropriate time or processed individually.

In another preferred embodiment, a further application is provided tobioreaction systems wherein the biological product is expressed insidethe cell as an inclusion body. The bioreactor is operated as describedabove but instead of monitoring the concentration of biological product,the optical density of the biological culture is monitored. When apredetermined optical density is reached, the bioreaction process isstopped, the cells lysed chemically and the resultant inclusion bodiessolubilized, all inside the bioreactor. Once a suitable solubilizationof the inclusion body has been achieved, an appropriate mixture ofresins is added to bind the solubilized inclusion bodies.

The rest of the method is then followed for the separation of thenutrient medium, detachment of solubilized inclusion bodies from theresin and further purification. It is further noted that there may notbe a need for further filtration to remove cells, as they would all havebeen lysed. In some instances, the protein can be refolded within thebioreactor using appropriate refolding buffer after detaching thesolubilized inclusion bodies from the resin.

The bioreactor container offers a remarkable opportunity to extend theuse to refold proteins eliminating the need for operating anothervessel. It is almost ironic that in general practice, the volume of therefolding solution is generally equal to the nutrient medium, making thebioreactor and ideal choice for protein refolding.

In another preferred embodiment, a further utility of the instantinvention is provided wherein the operation of bioreactor as describedabove produces a mixture of the biological product and the resin andthis complex can be directly loaded into chromatography purificationcolumns avoiding another cumbersome and time-consuming step.

Generally, the invention provides bioreactors and methods, which areuniversal in the sense that the invention is suitable and adaptable forprocessing a variety of compositions, including both biologic andnon-biologic components. Indeed, an inventive bioreactor designed foruse with mammalian cells, for example, may be used for culturingbacteria, allowing ease of manufacturing.

As used herein, the term “liquid” is intended to encompass compositions,which include biologic components as described herein.

Compositions comprising non-biologic components include, but are notlimited to, those which comprise microcarriers (e.g., polymer spheres,solid spheres, gelatinous particles, microbeads, and microdisks that canbe porous or non-porous), cross-linked beads (e.g., dextran) chargedwith specific chemical groups (e.g., tertiary amine groups), 2Dmicrocarriers including cells trapped in nonporous polymer fibers, 3Dcarriers (e.g., carrier fibers, hollow fibers, multicartridge reactors,and semi-permeable membranes that can comprising porous fibers),microcarriers having reduced ion exchange capacity, cells, capillaries,and aggregates (e.g., aggregates of cells).

The biological components that may be processed in accordance with theinvention are described in the paragraphs which follow and include, butare not limited to, cell cultures derived from sources such as animals(e.g., hamsters, mice, pigs, rabbits, dogs, fish, shrimp, nematodes, andhumans), insects (e.g., moths and butterflies), plants (e.g., algae,corn, tomato, rice, wheat, barley, alfalfa, sugarcane, soybean, potato,lettuce, lupine, tobacco, reapeseed (canola), sunflower, turnip, beetcane molasses, seeds, safflower, and peanuts), bacteria, fungi, andyeast.

Illustrative animal cells include Chinese hamster ovary (CHO), mousemyeloma, M0035 (NSO cell line), hybridomas (e.g., B-lymphocyte cellsfused with myeloma tumor cells), baby hamster kidney (BHK), monkey COS,African green monkey kidney epithelial (VERO), mouse embryo fibroblasts(NIH-3T3), mouse connective tissue fibroblasts (L929), bovine aortaendothelial (BAE-1), mouse myeloma lymphoblastoid-like (NSO), mouseB-cell lymphoma lymphoblastoid (WEHI 231), mouse lymphoma lymphoblastoid(YAC 1), mouse fibroblast (LS), hepatic mouse (e.g., MC/9, NCTC clone1469), and hepatic rat cells (e.g., ARL-6, BRL3A, H4S, Phi 1 (from Fu5cells)).

Illustrative human cells include retinal cells (PER-C6), embryonickidney cells (HEK-293), lung fibroblasts (MRC-5), cervix epithelialcells (HELA), diploid fibroblasts (WI38), kidney epithelial cells (HEK293), liver epithelial cells (HEPG2), lymphoma lymphoblastoid cells(Namalwa), leukemia lymphoblastoid-like cells (HL60), myelomalymphoblastoid cells (U 266B1), neuroblastoma neuroblasts (SH-SY5Y),diploid cell strain cells (e.g., propagation of poliomyelitis virus),pancreatic islet cells, embryonic stem cells (hES), human mesenchymalstem cells (MSCs, which can be differentiated to osteogenic,chondrogenic, tenogenic, myogenic, adipogenic, and marrow stromallineages, for example), human neural stem cells (NSC), human histiocyticlymphoma lymphoblastoid cells (U937), and human hepatic cells such asWRL68 (from embryo cells), PLC/PRF/5 (i.e., containing hepatitis Bsequences), Hep3B (i.e., producing plasma proteins: fibrinogen,alpha-fetoprotein, transferrin, albumin, complement C3 and/oralpha-2-macroglobulin), and HepG2 (i.e., producing plasma proteins;prothrombin, antithrombin III, alpha-fetoprotein, complement C3, and/orfibrinogen).

Cells from insects (e.g., baculovirus and Spodoptera frugiperda ovary(Sf21 cells produce Sf9 line)) and cells from plants or food, may alsobe cultured in accordance with the invention. Cells from sources such asrice (e.g., Oryza sativa, Oryza sativa cv Bengal callus culture, andOryza sativa cv Taipei 309), soybean (e.g., Clycine max cv Williams 82),tomato (Lycopersicum esculentum cv Seokwang), and tobacco leaves (e.g.,Agrobacterium tumefaciens including Bright Yellow 2 (BY-2), Nicotianatabacum cv NT-1, N. tabacum cv BY-2, and N. tabacum cv Petite HavanaSR-1) are illustrative examples.

Bacteria, fungi, or yeast may also be cultured in accordance with theinvention. Illustrative bacteria include Salmonella, Escherichia coli,Vibrio cholerae, Bacillus subtilis, Streptomyces, Pseudomonasfluorescens, Pseudomonas putida, Pseudomonas sp, Rhodococcus sp,Streptomyces sp, and Alcaligenes sp. Fungal cells can be cultured fromspecies such as Aspergillus niger and Trichoderma reesei, and yeastcells can include cells from Hansenula polymorpha, Pichia pastoris,Saccharomyces cerevisiae, S. cerevisiae crossed with S. bayanus, S.cerevisiae crossed with LAC4 and LACI-2 genes from K. lactis, S.cerevisiae crossed with Aspergillus shirousamii, Bacillus subtilis,Saccharomyces diastasicus, Schwanniomyces occidentalis, S. cerevisiaewith genes from Pichia stipitis, and Schizosaccharomyces pombe.

A variety of different products may also be produced in accordance withthe invention. Illustrative products include proteins (e.g., antibodiesand enzymes), vaccines, viral products, hormones, immunoregulators,metabilites, fatty acids, vitamins, drugs, antibiotics, cells, andtissues. Non-limiting examples of proteins include human tissueplasminogen activators (tPA), blood coagulation factors, growth factors(e.g., cytokines, including interferons and chemokines), adhesionmolecules, Bcl-2 family of proteins, polyhedrin proteins, huyman serumalbumin, scFv antibody fragment, huyman erythropoietin, mouse monoclonalheavy chain 7, mouse IgG_(2b/k), mouse IgG1, heavy chain mAb, Bryondin1, human interleukin-2, human interleukin-4, ricin, humanα1-antitrypisin, biscFv antibody fragment, immunoglobulins, humangranulocyte, stimulating factor (hGM-CSF), hepatitis B surface antigen(HBsAg), human lysozyme, IL-12, and mAb against HBsAg. Examples ofplasma proteins include fibrinogen, alpha-fetoprotein, transferrin,albumin, complement C3 and alpha-2-macroglobulin, prothrombin,antithrombin III, alpha-fetoprotein, complement C3 and fibrinogen,insulin, hepatitis B surface antigen, urate oxidase, glucagon,granulocyte-macrophage colony stimulating factor, hirudin/desirudin,angiostatin, elastase inhibitor, endostatin, epidermal growth factoranalog, insulin-like growth factor-1, kallikrein inhibitor,α1-antitrypsin, tumor necrosis factor, collagen protein domains (but notwhole collagen glycoproteins), proteins without metabolic byproducts,human albumin, bovine albumin, thrombomodulin, transferrin, factor VIIIfor hemophilia A (i.e., from CHO or secreted alkaline phosphatase,aprotinin, histamine, leukotrienes, IgE receptors,N-acetylglucosaminyltransferase-III, and antihemophilic factor VIII.

Enzymes may be produced from a variety of sources using the invention.Non-limiting examples of such ensymes include YepACT-AMY-ACT-X24 hybridensyme from yeast, Aspergillus oryzae α-amylase, xylanases, urokinase,tissue plasminogen activator (rt-PA), bovine chymosin,glucocerebrosidase (therapeutic enzyme for Gaucher's disease, from CHO),lactase, trypsin, aprotinin, huyman lactoferrin, lysozyme, andoleosines.

Vaccines also may be produced using the invention. Non-limiting examplesinclude vaccines for prostate cancer, human papilloma virus, viralinfluenza, trivalent hemagglutinin influenze, AIDS, HIV, malaria,anthrax, bacterial meningitis, chicken pox, cholera, diphtheria,haemophilus influenze type B, hepatitis A, hepatitis B, pertussis,plague, pneumococcal pneumonia, polio, rabies, human-rabies, tetanus,typhoid fever, yellow fever, veterinary-FMD, New Castle's Disease, footand mouth disease, DNA, Venezuelan equine encephalitis virus, cancer(colon cancer) vaccines (i.e., prophylactic or therapeutic), MMR(measles, mumps, rubells), yellow fever, Haemophilus influenzae (Hib),DTP (diphtheria and tetanus vaccines, with pertussis subunit), vaccineslinked to polysaccharides (e.g., Hib, Neisseria meningococcus),Staphylococcus pneumoniae, nicotine, multiple sclerosis, bovinespongiform encephalopathy (mad cow disease), IgGI (phosphonate ester),IgM (neuropeptide hapten), SIgA/G (Streptococcus mutans adhesin),scFv-bryodin 1 immunotoxin (CD-40), IgG (HSV), LSC(HSV), Norwalk virus,human cytomegalovirus, rotavirus, respiratory syncytial virus F,insulin-dependent autoimmune mellitus diabetes, diarrhea, rhinovirus,herpes simplex virus, and personalized cancer vaccines, e.g., forlymphoma treatment (i.e., in injectable, oral, or edible forms).Recombinant subunit vaccines also may be produced, such as hepatitis Bvirus envelope protein, rabies virus glycoprotein, E. coli heat labileenterotoxin, Norwalk virus capsid protein, diabetes autoantigen, choleratoxin B subunit, cholera toxin B and dA2 subunits, rotavirus enterotoxinand enterotoxigenic E. coli, fimbrial antigen fusion, and porcinetransmissible gastroenteritis virus glycoprotein S.

Viral products also may be produced. Non-limiting examples of viralproducts include sindbis, VSV, oncoma, hepatitis A, channel cat fishvirus, RSV, corona virus, FMDV, rabies, polio, reo virus, measles, andmumps.

Hormones also may be produced using the invention. Non-limiting examplesof hormones include growth hormone (e.g., human growth hormone (hGH) andbvine growth hormone), growth factors, beta and gamma interferon,vascular endothelial growth factor (VEGF), somatostatin,platelet-derived growth factor (PDGF), follicle stimulating hormone(FSH), luteinizing hormone, human chorionic hormone, and erythropoietin.

Immunoregulators also may be produced. Non-limiting examples ofimmunoregulators include interferons (e.g., beta-interferon (formultiple sclerosis), alpha-interferon, and gamma-interferon) andinterleukins (such as IL-2).

Metabolites (e.g., shikonin and paclitaxel) and fatty acids (i.e.,including straight-chain (e.g., adipic acid, Azelaic acid, 2-hydroxyacids), branched-chain (e.g., 10-methyl octadecanoic acid and retinoicacid), ring-including fatty acids (e.g., coronaric acid and lipoicacid), and complex fatty acids (e.g., fatty acyl-CoA)) also may beproduced.

The containers useful in the various embodiments of the invention may beof any size suitable for containing a liquid. For example, the containermay have a volume between 1-40 L, 40-100 L, 100-200 L, 200-300 L,300-500 L, 500-750 L, 750-1,000 L, 1,000-2,000 L, 2,000-5,000 L, or5,000-10,000 L. In some instances, the container has a volume greaterthan 1 L, or in other instances, greater than 10 L, 20 L, 40 L, 100 L,200 L, 500 L, or 1,000 L. Volumes greater than 10,000 L are alsopossible. Preferably, the container volume will range between about 1 Land 1000 L, and more preferably between about 5 L and 500 L, and evenmore preferably between 5 L and 200 L.

The components of the bioreactors and other devices described hereinwhich come into contact with the culture medium or products providedthereby desirably comprise biocompatible materials, more desirablybiocompatible polymers, and are preferably sterilizable.

It should also be understood that many of the components describedherein also are desirably flexible, e.g., the containers desirablycomprise flexible biocompatible polymer containers (such as collapsiblebags), with the conduits also desirably comprising such biocompatiblepolymers. The flexible material is further desirably one that is USPClass VI certified, e.g., silicone, polycarbonate, polyethylene, andpolypropylene. Non-limiting examples of flexible materials includepolymers such as polyethylene (e.g., linear low density polyethylene andultra low density polyethylene), polypropylene, polyvinylchloride,polyvinyldichloride, polyvinylidene chloride, ethylene vinyl acetate,polycarbonate, polymethacrylate, polyvinyl alcohol, nylon, siliconerubber, other synthetic rubbers and/or plastics. If desired, portions ofthe flexible container may comprise a substantially rigid material suchas a rigid polymer (e.g., high density polyethylene), metal, and/orglass.

Desirably the containers comprise biocompatible materials, moredesirably biocompatible polymers. When collapsible containers areselected for use, the container may be supported by or may line an innersurface of a support structure, e.g., the outer support housing havingcontainer-retaining sidewalls. However, the invention may be practicedusing non-collapsible or rigid containers or conduits.

The containers may have any thickness suitable for retaining the culturemedium there within, and may be designed to have a certain resistance topuncturing during operation or while being handled. For example, thewalls of a container may have a total thickness of less than or equal to250 mils (1 mil is 25.4 micrometers), less than or equal to 200 mils,less than or equal to 100 mils, less than or equal to 70 mils (1 mil is25.4 micrometers), less than or equal to 50 mils, less than or equal to25 mils, less than or equal to 15 mils, or less than or equal to 10mils. In certain embodiments, the container may include more than onelayer of material that may be laminated together or otherwise attachedto one another to impart certain properties to the container. Forinstance, one layer may be formed of a material that is substantiallyoxygen impermeable. Another layer may be formed of a material to impartstrength to the container. Yet another layer may be included to impartchemical resistance to fluid that may be contained in the container.

It thus should be understood that a container may be formed of anysuitable combinations of layers. The container (e.g., collapsible bag)may include, for example, 1 layer, greater than or equal to 2 layers,greater than or equal to 3 layers, or greater than equal to 5 layers ofmaterial(s). Each layer may have a thickness of, for example, less thanor equal to 200 mils, less than or equal to 100 mils, less than or equalto 50 mils, less than or equal to 25 mils, less than or equal to 15mils, less than or equal to 10 mils, less than or equal to 5 mils, orless than or equal to 3 mils, or combinations thereof.

In addition, the container preferably is seamless in order to improveits strength and avoid deposition of growing cells in the media.

All or portions of the container also are desirably translucent, or moredesirably transparent, to allow viewing of contents inside thecontainer. The latter is preferred when it is desirable to irradiate theculture medium within the container.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Prior Art

The instant invention is a type of separative bioreactor. In the pastsubstantial progress has been made in membrane bioreactors (MBR) thathad the ability to separate the products within the bioreactors. The MBRprocess was introduced by the late 1960s, as soon as commercial scaleultrafiltration (UF) and microfiltration (MF) membranes were available.The original process was introduced by Dorr-Olivier Inc. and combinedthe use of an activated sludge bioreactor with a cross-flow membranefiltration loop. The flat sheet membranes used in this process werepolymeric and featured pore sizes ranging from 0.003 to 0.01 μm.Although the idea of replacing the settling tank of the conventionalactivated sludge process was attractive, it was difficult to justify theuse of such a process because of the high cost of membranes, loweconomic value of the product (tertiary effluent) and the potentialrapid loss of performance due to membrane fouling. As a result, thefocus was on the attainment of high fluxes, and it was thereforenecessary to pump the mixed liquor suspended solids (MLSS) at highcross-flow velocity at significant energy penalty (of the order 10kWh/m3 product) to reduce fouling. Due to the poor economics of thefirst generation MBRs, they only found applications in niche areas withspecial needs like isolated trailer parks or ski resorts for example.

The breakthrough for the MBR came in 1989 with the idea of Yamamoto andco-workers to submerge the membranes in the bioreactor. Until then, MBRswere designed with the separation device located external to the reactor(side-stream MBR) and relied on high transmembrane pressure (TMP) tomaintain filtration. With the membrane directly immersed into thebioreactor, submerged MBR systems are usually preferred to sidestreamconfiguration, especially for domestic wastewater treatment. Thesubmerged configuration relies on coarse bubble aeration to producemixing and limit fouling. The energy demand of the submerged system canbe up to 2 orders of magnitude lower than that of the sidestream systemsand submerged systems operate at a lower flux, demanding more membranearea. In submerged configurations, aeration is considered as one of themajor parameter on process performances both hydraulic and biological.Aeration maintains solids in suspension, scours the membrane surface andprovides oxygen to the biomass, leading to a better biodegradability andcell synthesis.

The other key steps in the recent MBR development were the acceptance ofmodest fluxes (25% or less of those in the first generation), and theidea to use two-phase bubbly flow to control fouling. The loweroperating cost obtained with the submerged configuration along with thesteady decrease in the membrane cost encouraged an exponential increasein MBR plant installations from the mid 90s. Since then, furtherimprovements in the MBR design and operation have been introduced andincorporated into larger plants. While early MBRs were operated at solidretention times (SRT) as high as 100 days with mixed liquor suspendedsolids up to 30 g/L, the recent trend is to apply lower solid retentiontimes (around 10-20 days), resulting in more manageable mixed liquorsuspended solids (MLSS) levels (10-15 g/L). Thanks to these newoperating conditions, the oxygen transfer and the pumping cost in theMBR have tended to decrease and overall maintenance has been simplified.There is now a range of MBR systems commercially available, most ofwhich use submerged membranes although some external modules areavailable; these external systems also use two-phase flow for foulingcontrol. Typical hydraulic retention times (HRT) range between 3 and 10hours. In terms of membrane configurations, mainly hollow fiber and flatsheet membranes are applied for MBR applications.

Despite the more favorable energy usage of submerged membranes, therecontinued to be a market for the side stream configuration, particularlyin industrial applications. For ease of maintenance the side streamconfiguration can be installed at low level in a plant building.Membrane replacement can be undertaken without specialist equipment, andintensive cleaning of individual banks can be undertaken during normaloperation of the other banks and without removing the membranes modulesfrom the installation.

As a result research continued with the side stream configuration,during which time it was found that full-scale plants could be operatedwith higher fluxes. This has culminated in recent years with thedevelopment of low energy systems which incorporate more sophisticatedcontrol of the operating parameters coupled with periodic back washes,which enable sustainable operation at energy usage as low as 0.3 kWh/m3product.

Argonne scientists (www.anl.gov) recently used electrical force totransport organic acids away from the biocatalyst across an ion-exchangemembrane and into a concentrate chamber, very similar to normalmetabolism processes for handling acids. To provide the electricity in acost efficient fashion, researchers turned to electrodeionization (EDI).EDI is an established commercial technology for producing high-puritywater. Previously, Argonne scientists modified EDI so that it could beused for desalination of chemical and agricultural products. Toaccomplish this, researchers molded loose ion exchange resin beads intoa porous resin wafer, enabling the capture of charge salts and acids atdilution levels with high-energy efficiency and significantly reducedwaste streams compared to conventional processing. This became the basisfor the Argonne's separative bioreactor.

Researchers also realized that although direct enzyme immobilization onmembranes provided excellent product separations, insufficient enzymedensity limited the overall performance. In order to increase thedensity, the scientists integrated enzyme immobilization technology intothe porous resin wafer and created a material that can efficientlyproduce and remove organic acids. As Argonne designed its separativebioreactor, researchers incorporated enzyme capture resin beads into theresin wafer. Sugars were converted by the immobilized biocatalyst to thetarget acids, and the product was electrically transported into aconcentrate channel. This resulted in reactions occurring withoutbuffering or neutralization. Argonne's immobilization technology alsoallows in-situ stripping and replacement of degraded enzymes withoutdisassembling the system.

However, every type of membrane separative bioreactor disclosed utilizeda similar principle of forcing a biological product across a membrane.The instant invention differs significantly by roviding a device capableof containing a resin capable of binding the target biological product,the membrane holding the resin has no specific function except to keepthe resin separated form the bulk liquid in the bioreactors and also toprevent larger scale organisms or cells to contact the resin. Theseparation function in the instant invention is provided by anon-specific, non-electrically driven reaction.

The prior art on the design and operation of separative bioreactors issilent on the concept of instant invention. The main references toseparative bioreactors of use in biological sciences appear as U.S.patent application Ser. No. 10/993,642 filed 19 Nov. 2004 wherein aseparative bioreactor is disclosed. Accordingly, it is a separativebioreactor, comprising an anode and a cathode, a plurality of reactionchambers each having an inlet and an outlet and each including a poroussolid ion exchange wafer having ion-exchange resins, each of thereaction chambers being interleaved between a cation exchange membraneand an anion exchange membrane or between either a cation or an anionexchange membrane and a bipolar exchange membrane, a plurality ofproduct chambers each having an inlet and an outlet and separated fromone of the reaction chambers by either a cation or an anion exchangemembrane, recirculation mechanism for transporting material between thereaction chamber inlets and outlets and for transporting product betweenthe product chamber inlets and outlets, and mechanism for supplying anelectric potential between the anode and the cathode causing ions to betransported between chambers, whereby counterions retained or producedin each of the reaction chambers during the production of an ionizableorganic product including product ions combine with oppositely chargedions to form molecules some or all of which are transported to reactionchamber inlets while product ions are transported into an adjacentproduct chamber to combine with oppositely charged ions to form productin a product stream exiting the product chamber outlets continuouslyrecirculated to the product chamber inlets to increase the concentrationof product in the product stream. None of the features described in thisapplication are material to the instant invention and none of theessential features of the instant inventions are disclosed in thisapplication.

The U.S. Pat. No. 7,306,934 issued 11 Dec. 2007 discloses a porous solidion exchange wafer for immobilizing biomolecules, said wafer comprisinga combination of an biomolecule capture-resin containing a transitionmetal cation of +2 valence and an ion-exchange resin. The patent furtherdiscloses a separative bioreactor, comprising an anode and a cathode, aplurality of reaction chambers at least some being formed from a poroussolid ion exchange wafers having a combination of art biomoleculecapture-resin and an ion-exchange resin and having a geneticallyengineered tagged biomolecule immobilized on said biomolecule captureresin, each of said porous solid ion exchange wafers being interleavedbetween a cation exchange membrane and an anion exchange membrane, andmechanism for supplying an electric potential between the anode and thecathode. The instant invention does not rely on any features disclosedin this patent, nor any features of the instant invention are recited inthis patent.

The U.S. Pat. No. 7,799,548 issued 21 Sep. 2010 is for a method of insitu stripping a genetically tagged biomolecule from a porous solid ionexchange wafer in a bioreactor, the wafer having a combination of abiomolecule capture-resin and an ion-exchange resin forming a chargedcapture resin within the wafer and having a genetically taggedbiomolecule immobilized on said biomolecule capture-resin, comprisingcontacting the porous solid ion exchange wafer in the bioreactor with astripping fluid at a temperature and for a time sufficient to strip atleast some of the genetically tagged biomolecule therefrom. This patentadditionally claims method of in situ stripping a genetically taggedbiomolecule from a porous solid ion exchange wafer in a bioreactor andthereafter regenerating a genetically tagged biomolecule onto the poroussolid ion exchange wafer, the wafer having a combination of abiomolecule capture-resin and an ion-exchange resin forming a chargedcapture resin within the wafer and having a genetically taggedbiomolecule immobilized on said biomolecule capture-resin thereon,comprising contacting the porous solid ion exchange wafer in thebioreactor with a stripping fluid at a temperature and for a timesufficient to strip at least some of the genetically tagged biomoleculestherefrom, and thereafter contacting the stripped porous solid ionexchange wafer in the bioreactor with an effective amount of agenetically tagged biomolecules at a temperature and for a timesufficient to immobilize genetically tagged biomolecules on the chargedcapture resin. The instant invention does not rely on any disclosuresmade in this patent nor are any of the essential features of the instantinvention disclosed in this patent.

The U.S. Pat. No. 7,141,154 issued 28 Nov. 2006 discloses a method ofcontinuously making an organic ester from a lower alcohol and an organicacid, comprising, introducing an organic acid or an organic salt intoand/or producing an organic acid or an organic salt in anelectrodeionization (EDI) stack having an anode and a cathode and aplurality of reaction chambers each formed from a porous solid ionexchange resin wafer interleaved between anion exchange membranes or ananion exchange membrane and a cation exchange membrane or an anionexchange membrane and a bipolar exchange membrane, providing mechanismfor establishing an electric potential between the EDI anode andcathode, wherein at least some reaction chambers are esterificationchambers and/or bioreactor chambers and/or chambers containing anorganic acid or salt, whereby an organic acid or organic salt present inthe EDI stack disassociates into a cation and an anion with the anionmigrating into an associated esterification chamber through an anionexchange membrane if required and reacting with a lower alcohol in theesterification chamber fo form an organic ester and water with at leastsome of the water splitting into a proton and a hydroxyl anion with atleast some of the hydroxyl anion migrating to an adjacent chamber, saidmigration of ions being facilitated by establishing an electricpotential across the EDI anode and cathode. The patent additionallydiscloses an apparatus for manufacturing an organic ester, comprising anelectrodeionization (EDI) stack having an anode and a cathode and aplurality of reaction chambers each formed from a porous solid ionexchange resin wafer interleaved between anion exchange membranes or ananion exchange membrane and either a cation exchange membrane or abipolar membrane, mechanism for establishing an electrical potentialbetween said EDI anode and said cathode, at least some of said reactionchambers being esterification chambers or esterification chambersseparated from an adjacent bioreactor chamber by an anion exchangemembrane and/or an acid/base capture chamber, said bioreactor chamberseach containing an ion exchange resin wafer capable of froming anorganic acid or salt from an ionizable fluid flowing therein, saidesterification chambers each containing an ion exchange resin wafercapable of forming an organic ester and water from a lower alchol and ananion of an organic acid or salt, a source of anions supplied directlyto said esterification chambers or supplied from adjacent chambers, anda supply of lower alcohol to said esterification chambers, whereby whena potential is established across said EDI anode and cathode at leastsome hydroxyl anions in said esterification chambers from watersplitting migrate across said anion exchange membranes to adjacentchambers to drive the reaction to continuously produce an organic ester.None of the features disclosed in this patent are material to theinstant invention and none of the essential features of the instantinvention are disclosed or taught in this patent.

In summary, the prior art disclosed above teaches the use of poroussolid ion exchange wafer for immobilizing biomolecules, said wafercomprising a combination of an biomolecule capture-resin containing atransition metal cation of +2 valence; it also teaches a separativebioreactor, comprising an anode and a cathode, a plurality of reactionchambers at least some being formed from a porous solid ion exchangewafers (above) having a combination of art biomolecule capture-resin andan ion-exchange resin and having a genetically engineered taggedbiomolecule immobilized on said biomolecule capture resin, each of saidporous solid ion exchange wafers being interleaved between a cationexchange membrane and an anion exchange membrane, and mechanism forsupplying an electric potential between the anode and the cathode.

The instant invention is significantly different from the separativebioreactor taught above. First, the instant invention does not requireuse of electrodes, or resins with a transition cation of +2 valence orimmobilized metal ion affinity chromatography. The use of EDI(electrodeionization) and specific use of tags and limited nature ofsolvents to remove the captured biological products, mainly enzymes,makes this patent treaching distinctly different from the instantinvention. In addition, and most significantly, the prior art can not beused with the preferred embodiment of the instant invention whereinflexible bioreactors are taught.

Moreover, the prior art requires additional hardware that addssubstantial cost to the processing of manufacturing biological productswhile the instant invention combines several processes into one withoutadding any new cost element. The prior art is also specific to certaintypes of molecules while the instant invention is generic to every typeof biological product.

I claim:
 1. A harvesting device for capturing a biological productcomprising at least one container with at least one external surface andan inner volume to hold at least one ligand or resin capable of bindinga biological product and the surface having a plurality of pores.
 2. Theharvesting device according to claim 1, wherein the container is aflexible pouch.
 3. The harvesting device according to claim 1, whereinthe container comprises a plastic, nylon, metallic mesh, or a compositematerial.
 4. The harvesting device according to claim 1, wherein thesize of pores is between 0.2 and 30 microns.
 5. The harvesting deviceaccording to claim 1, wherein the size of pores is between 1 and 5microns.
 6. The harvesting device according to claim 1, wherein theresin or ligand is an ionic-exchange resin, a hydrophobic resin, astimuli responsive polymer, an affinity resin, Protein A-coated magneticbeads, Protein-G magnetic beads, a dual affinity polypeptide, Q Hypercelsorbant, S Hypercel sorbent, 4-mercaptoethylpyridine (MEP), Protein G,countercurrent chromatography media, a peptide ligand, an Fe-receptormimetic ligand, sn inorganic ligand, a synthetic dye, or a single-domaincamel-derived anti-IgG antibody.
 7. The harvesting device according toclaim 1, wherein the container comprises a plurality of resins.
 8. Theharvesting device according to claim 1, wherein there is a plurality ofcontainers.
 9. The harvesting device according to claim 1, wherein theharvesting device is disposed inside a bioreactor.
 10. The harvestingdevice according to claim 9, wherein the device is used at the end of abioreactor operation cycle.
 11. The harvesting device according to claim9, wherein the device is used during a bioreactor operation cycle. 12.The harvesting device according to claim 1, wherein the device is usedto capture a biological product from a nutrient media comprising abiological culture contained in a bioreactor.
 13. The harvesting deviceaccording to claim 1, wherein the biological product is produced usingbacteria, yeast, hybrodomas, baculoviruses, animal cells, mammaliancells or plant cells.
 14. The harvesting device according to claim 1,wherein the biological product comprises solubilized inclusion bodies,small proteins, enamel matrix proteins, fusion proteins, tag proteins,hormones, parathyroid hormones, growth hormones, gonadotripins, insulin,ACTH, prolactin, placental lactogen, melanocyte stimulating hormone,thyrotropin, calcitonin, enkephalin, angiotensin, cytokines human serumalbumin, bovine serum albumin, ovalbumin, glucose isomerase, α-amylase,endo-β-glucanase, growth hormone (GH), IGF-1, IGF-2, PTH, PGE₂, TGF-β,TGF-α, bEGF, EGF, PDGF-AB, PDGF-BB, osteoprotegerin (OPG), osteopontin(OP), FGF-1, FGF-2, thyroid hormone, BMP-2, BMP-3, BMP-4, BMP-6, BMP-7,VEGF, L25(OH)₂,vitamin D₃, caclitonin, IFN-alpha, IFN-beta, IFN-gamma,OCN (osteocalcin), ON (osteonectin), OP-1 (osteogenic protein-1), NGF,collagen, fibronectin, fibrinogen, thrombin, factor XIII, a recombinantprotein, a recombinant antibody, or a recombinant peptide.